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	crtN-F    5’gctagCTCGAGttaGGATCCtcagtaactggctgacaagcct3’		crtN-F    5’gctagCTCGAGttaGGATCCtcagtaactggctgacaagcct3’
	crtN-R    3’ccaaaAGATCTgtgaaacatacagcaaaaaacctgggt5’		crtN-R    3’ccaaaAGATCTgtgaaacatacagcaaaaaacctgggt5’
-	bchB-F    5'ttaaGAATTCaagaaggagatatacatATGGGCGGAAGCGGGGTGGCTGGA	+	bchB-F    5'ttaaGAATTCaagaaggagatatacatATGGGCGGAAGCGGGGTGGCTGGA3'
	bchB-R    3'ttaGGATCCccgttcgccttggtttgacttact5'		bchB-R    3'ttaGGATCCccgttcgccttggtttgacttact5'
-	bchE-F    ccaaagatctAAGAAGGAGATATACATatgcgcatactgatgatcca	+	bchE-F    5'ccaaagatctAAGAAGGAGATATACATatgcgcatactgatgatcca3'
-	bchE-R    gctagCTCGAGtattttcatcatgcctctcgt	+	bchE-R    3'gctagCTCGAGtattttcatcatgcctctcgt5'
-	bchI-F    ttaaGAATTCaagaaggagatatacatATGACGGAAGTGCAAAACAAT	+	bchI-F    5'ttaaGAATTCaagaaggagatatacatATGACGGAAGTGCAAAACAAT3'
-	bchI-R    ttaGGATCCtcggggctgagaaggcgggagca	+	bchI-R    3'ttaGGATCCtcggggctgagaaggcgggagca5'
-	bchL-F    ttaaGAATTCaagaaggagatatacatATGATCATCGCGGTCTACGGA	+	bchL-F    5'ttaaGAATTCaagaaggagatatacatATGATCATCGCGGTCTACGGA3'
-	bchL-R    ttaGGATCCttgggcagaaggtgtggaagca	+	bchL-R    3'ttaGGATCCttgggcagaaggtgtggaagca5'
-	bchM-F    ttaaGAATTCaagaaggagatatacatATGGCAAACGAAGTAAATTC	+	bchM-F    5'ttaaGAATTCaagaaggagatatacatATGGCAAACGAAGTAAATTC3'
-	bchM-R    ttaaGGATCCtctcttcggcttaatttccaacag	+	bchM-R    3'ttaaGGATCCtctcttcggcttaatttccaacag5'
-	bchN-F    accgaattcAAGAAGGAGATATACATatggaaagggtcgaacgggaaaac	+	bchN-F    5'accgaattcAAGAAGGAGATATACATatggaaagggtcgaacgggaaaac3'
-	bchN-R    attaggatccTCATTCCAGCCACCCCGCTT	+	bchN-R    3'attaggatccTCATTCCAGCCACCCCGCTT5'
	'''PCR'''		'''PCR'''

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Revision as of 05:50, 27 October 2007

Contents 1 Materials 2 Methods 3 Results 4 Discussion

Materials

Methods

Primer Design

Biobricking of bch-gene cluster and crtN gene.

Gene:

bchB

bchE

bchI

bchL

bchM

bchN

[[crtNgene]]

crtN

PCR of crtN on Heliobacillus mobilis G/C content on annealing region

crtN-F 40% BglII CrtN-R 50% BamHI and XhoI

Primers

crtN-F     5’gctagCTCGAGttaGGATCCtcagtaactggctgacaagcct3’
crtN-R     3’ccaaaAGATCTgtgaaacatacagcaaaaaacctgggt5’
bchB-F     5'ttaaGAATTCaagaaggagatatacatATGGGCGGAAGCGGGGTGGCTGGA3'
bchB-R     3'ttaGGATCCccgttcgccttggtttgacttact5'
bchE-F     5'ccaaagatctAAGAAGGAGATATACATatgcgcatactgatgatcca3' 
bchE-R     3'gctagCTCGAGtattttcatcatgcctctcgt5'
bchI-F     5'ttaaGAATTCaagaaggagatatacatATGACGGAAGTGCAAAACAAT3'
bchI-R     3'ttaGGATCCtcggggctgagaaggcgggagca5'
bchL-F     5'ttaaGAATTCaagaaggagatatacatATGATCATCGCGGTCTACGGA3'   
bchL-R     3'ttaGGATCCttgggcagaaggtgtggaagca5'
bchM-F     5'ttaaGAATTCaagaaggagatatacatATGGCAAACGAAGTAAATTC3'
bchM-R     3'ttaaGGATCCtctcttcggcttaatttccaacag5'
bchN-F     5'accgaattcAAGAAGGAGATATACATatggaaagggtcgaacgggaaaac3'
bchN-R     3'attaggatccTCATTCCAGCCACCCCGCTT5'

PCR

  PCR of bch-? genes using - 20ng/ul pHM6
  1ul pHM6 
  1ul dNTP
  5ul primer mix 
  0.5ul enzyme-phusion polymerase
  32.5ul H20
  50ul total
   

Digestions

  6 ul bch insert
  8ul pET29bEBBX
  2ul ligase buffer
  1ul ligase 
  3ul H20
  20ul total 

Dephosphorylation

  50 ul pBR3222
  6 ul phosphatase buffer
  1 ul phosphatase
  3 ul H20
  Incubate 30" -1hr 
  run gel

Ligations (3hrs @ R.T)

Results

Sequencing

Discussion